Composition for diagnostic reagents

ABSTRACT

A fixative composition for histological, cytological, immunological and proteinaceous preparations comprising a mixture of pyrrolid-2-one, a polyol, a urea and a zinc salt of a non-oxidizing organic or inorganic acid. Also disclosed are devices utilizing such compositions.

CROSS REFERENCE

This is a divisional of Ser. No. 345,589 filed on Feb. 4, 1982, now U.S.Pat. No. 4,493,821.

DETAILED DESCRIPTION

This invention pertains to a fixative and preservative compositions forhistological, cytological, immunological and proteinaceous preparationsand to novel devices and test systems made possible by the uniqueproperties of the composition.

The use of fixatives to preserve histological and cytologicalpreparations is, of course, well known and widely practiced. Invariablythese are liquid preparations which are applied to the sample or inwhich the sample is immersed. Lerner et al. disclose in U.S. Pat. No.3,546,334 a cytological fixative solution of a polyalkylene glycol,water, a C-1 to C-10 alcohol and a ketone which is sprayed on to a slideon which has been previously placed a smear of body cells. According tothe disclosure, alcohol, water and ketone evaporate leaving a protectivefilm of the polyethylene glycol over the smear.

Westlake et al. in U.S. Pat. No. 3,997,656 describe a histologicalstaining method in which tissue is first immersed in an aqueous fixingsolution of trichloroacetic acid, zinc chloride and formaldehyde. A waxsuch as polyethylene glycol can be included in the fixing solution as alubricant and sealant.

Ehrenreich, in U.S. Pat. No. 3,389,052 enumerates various approaches tofixing cytological smears, all of which involve the application of aliquid fixative, often ethanol and ethyl ether. This reference alsodescribes the use of an improved composition in aerosol form of a loweralkanol, a mixture of a liquid and a solid polyethylene glycol andpropellant which is sprayed on the smear.

The present invention is a departure from these approaches in that itprovides a fixative which is preapplied to a slide or other test surfaceand which presents a substantially dry, non-fluid surface to which thesample is applied. Mere contact between the surface and the sampleeffects fixing and preservation so that only the act of transferring thesample to the test surface is required. The sample need not be disturbedas in the past by the separate application of fixative.

The advantages flowing from this development are numerous. Firstly,precoated slides can be provided to individual practitioners.Cytological smears then can be directly applied. The slide can besubjected to such diagnostic tests as indicated or maintained forsubsequent testing, without the need for additional fixing orpreservative operations.

In addition, there is no need to adhere to a rigid fixing protocol, asis often the case with liquid or spray fixatives. As a result, one canachieve greater uniformity of results, the pre-application of thefixative material being admirably suited to standardization.

Moreover, because of the essentially "dry" nature of the preparation andthe special advantages attendant to the use of plastic slides, it ispossible to send slides inexpensively through the mail. This not onlygreatly simplifies clinical screening but opens the opportunity forcentralized microscopic examination for practitioners who are widelyseparated geographically.

The fixative and preservative properties of the composition also permitsthe preparation of diagnostic devices utilizing unstable biologicalentities such as antigens and some antibodies.

Other advantages and objects of the invention will be apparent from thefollowing disclosure.

The basic composition comprises as its principal fixative andpreservative component a four component mixture of pyrrolid-2-one, apolyol, at least one urea and a zinc salt of a non-oxidizing organic orinorganic acid. Within this mixture, the relative proportions of thefour components can vary widely within certain broad ranges dependingupon the specific use for which the overall composition is intended.Generally the pyrrolid-2-one will comprise from about 10% to about 75%by weight of the mixture, the polyol will comprise from about 10% toabout 50% by weight of the mixture, the urea will comprise from about 1%to about 20% by weight of the mixture and the zinc salt will comprisefrom about 1 to about 10% by weight of the mixture. The actual relativepercentages will be selected from within the respective ranges so thattheir sum is 100% of the mixture.

The polyol is typically a polyalkylene glycol such as polyethyleneglycol or polypropylene glycol although other polyols such as glycerincan be use. One polyol which has proven to be most satisfactory ispolyethylene glycol of a molecular weight of about 200.

The zinc salt can be of any non-oxidizing acid including stronginorganic acids such as hydrochloric acid (i.e., zinc chloride) orweaker organic acids such as acetic acid (i.e. zinc acetate).

Without wishing to be bound by any theory, it appears that compositionsutilizing the foregoing mixture do not operate by a dehydrationprinciple. This is to be contrasted with previous fixative compositionsutilizing such materials as formaldehyde and alcohols. Rather themixture appears to "tie up" water molecules within the cell or material,maintaining both cellular and immunological characteristics.

As will be seen supra, the mixture can be presented in differentcompositional embodiments. For example, the mixture ingredients may bein the form of an aqueous solution. Depending upon the strength(concentration), such solutions can be utilized as fixative preparationsper se or as stock solutions for the preparation of precoated slides.For example, a 0.005% aqueous solution of a mixture of about 40 to 45%pyrrolid-2-one, about 40 to 45% polyethylene glycol, about 9 to 10% ureaand about 4 to 5% zinc acetate is a suitable preservative for UCGantigen carried on milk latex. Alternatively, a solution of the samemixture can be applied to a slide and allowed to dry, thereby presentinga fixative surface.

In addition to the foregoing components of the mixture, various othercomponents can also be present. The presence or absence of suchingredients will depend upon the the specific application. For example,immunological and proteinaceous preparations are often advantageouslyincluded on a carrier such as milk latex, coconut charcoal and the like.When these are to be combined with the basic mixture, it is desirable toinclude one or more colloid protective agents such, as for example, apolysaccharide derivative such as dextrin, carrageenan or an epihydrincross-linked sucrose, a polyvinyl acetate, acrylamide, acholine-cholesterol preparation or the like. Moreover in such colloidalpreparations, the addition of a small amount of a surfactant such as thealkylaryl polyether alcohols, sulfonates and the like also often can beadvantageous.

In instances where a greater degree of complexing fixation is desiredfor tissue and other proteinaceous preparations, the addition of a smallamount up to about 1% of a dialdehyde, such as glyoxal or glutaraldehydeis effective. Similarly other complexing agents, such as trichloroaceticacid can be added. Typically when present, trichloracetic acid isutilized in an amount corresponding to from about 10 to about 15% bytotal weight of the above-defined four component mixture.

For precoated slides it is often also desirable for adhesion of thefixed specimen to add a quantity of a film forming adjuvant such apolyvinyl alcohol, methyl ethyl cellulose, collagen or the like to theformulation. This can range from about 10% to about 20% by total weightof the above-defined four component mixture, generally added at theconclusion of other mixing operations.

In one embodiment of the present invention, a stock solution is utilizedto precoat slides or other test surfaces. These will consist of a basestrip of material which is non-absorbent to and insoluble in water, asfor example a conventional glass microscope slide or a plastic strip orslide. Suitable for plastics include polystyrene, polyacrylate,polymethacrylate, polyethylene, polypropylene, polycarbonates,polyvinylchloride, nitrocellulose and the like. The surface will carryat least one deposition area which can extend to the entire surface ormay be limited to one or more zones on the surface. Particularly in thecase of plastic, the zones may be defined by suitable indentations.

On each zone is carried the evaporative residue of the particularfixative and preservative composition. Thus, a solution of thecomposition is applied to the surface and allowed to evaporate withgentle heating, e.g. 40° C., until sufficient liquid (predominatelywater) is driven off and a subtantially dry but slightly tacky residueremains. Such a precoated slide or test surface is then ready to receivea histological, cytological, immunological or proteinaceous specimenwhich is fixed upon application.

One particularly surprising observation involves the utilization of thepresent compositions with plastic surfaces where it appears theprecoating tends to render the surface relatively impervious to powerfulorganic solvents utilized in clinical chemistry. Thus precoating apolystyrene or polymethacrylate surface as herein described results inthe coated surface resisting xylene, toluene and the like.

In a further embodiment of the invention, the precoated base stripserves as a diagnostic testing device. Thus there is combined with acomposition of the present invention a histological, cytological,immunological or proteinaceous diagnostic reagent system and thiscombination is then deposited on the base strip and dried as previouslydescribed. The reagent system includes conventional dyes and stains suchas methylene blue N, cresyl violet acetate, hematoxylin, and the like,as well as counterstains such as rosaniline, magenta II, picric acid andthe like (see generally U.S. Pat. Nos. 3,997,656 and 4,070,495), as wellas mitocondrial dyes such as actiflavin. More significantly, thediagnostic reagent system can include materials of a biological naturewhich ordinarily are not amenable to prior preparation and storage.Proteinaceous substances which are reactive in diagnostic immunologicalreactions can be combined with the composition of the invention. Typicalof these are immunological agents such as sera, pure antibodies andantigens. While slides or test surfaces containing certain driedimmunological components have been previously describe (see, e.g. U.S.Pat. No. 3,666,421), such systems have been limited to thoseimmunological or proteinaceous materials of high stability. In addition,these systems have required a plurality of zones to prevent prematurereaction, both physical and chemical, the contents of which must then bemixed in executing the particular test. Finally, such testing systems ordevices lack any degree of permanence after development.

The present system provides for the preservation of, and thus permitsthe utilization of, otherwise unstable biological reagents such asantigens. These include antigens to antibodies and antibody-likesubstances as RF (arthritis), rapid plasma reagin (syphilis), IgG, IM,β-UCG and the like. Similarly antibodies including antisera andmonoclonal antibodies can be incorporated in the composition and used toprecoat slides or diagnostic substrate surfaces. Upon drying, theparticular biological reagent is preserved but still retains itsbiological activity. In fact, the reagent can be preserved in a singlezone even in the presence of other reactive components which are notactivated until the sample is moistened, as by application of the testspecimen. After completion of the test, the system can be preserved dueto the remaining presence of the preserving and fixing composition, thuspermitting future and comparative study.

The following examples will serve to further typify the nature of theinvention but should not be construed as a limitation on the scopethereof, the invention being defined solely by the appended claims.

EXAMPLE 1

    ______________________________________                                        Ingredient              Amount                                                ______________________________________                                        pyrrolid-2-one          15     g.                                             urea                    1.65   g.                                             1,3-dimethyl urea       1.65   g.                                             zinc acetate            1.5    g.                                             polyethylene glycol (200 mw)                                                                          6.25   ml.                                            ______________________________________                                    

The ingredients are mixed with 500 ml. of distilled water and 80 g. of a1:1 mixture Gelvatol G 40/10 and G 20/60 are added. The final solutionis adjusted to pH 6.6 with aqueous sodium hydroxide. One part by weightof this preparation is combined with 9 parts by weight of charcoal/RPRantigen and 0.5 mg. of the mixture is then applied to a polystyreneslide and dried at 40° C. The single test zone is stable at roomtemperature and can be used in the serological detection of syphilis.RPR carbon particle antigen detects "reagin" an antibody-like substancepresent in sera of syphilitic persons (and occasionally in sera ofpersons with other acute or chronic conditions). When a specimencontains antibody, flocculation occurs with a coagglutination of thecarbon particles which appears as black clumps. This coagglutination canbe read macroscopically. Nonreactive specimens appear to have alight-gray color.

EXAMPLE 2

    ______________________________________                                        Ingredient              Amount                                                ______________________________________                                        98% pyrrolid-2-one      15.0   g.                                             urea                    3.5    g.                                             polyethylene glycol (200 mw)                                                                          6.25   ml.                                            zinc acetate            1.0                                                   ______________________________________                                    

The foregoing ingredients are mixed in 500 ml. of distilled water. Heatmay be applied as necessary to facilitate dissolution. This solution isapplied to glass slides and allowed to evaporate at moderatetemperatures (about 40° C.), producing a dry transparent surface.Cytological specimens placed on this surface are rapidly fixed andmaintain all cellular characteristics and stain ability.

EXAMPLE 3

To the basic four component mixture described in Example 2 are added thefollowing:

    ______________________________________                                        Ingredient        Amount (% of mixture)                                       ______________________________________                                        trichloroacetic acid                                                                            12.8%   (3.3 g.)                                            glyoxal           0.4%    (0.2 ml. of 40%                                                               solution)                                           ______________________________________                                    

When all ingredients are thoroughly mixed, 1.6 g. of polyvinyl alcohol(Gelvatol 40/10) are added. The composition is utilized as in Example 2.

EXAMPLE 4

To the composition of Example 3 are added two drops (about 0.06 ml) ofTriton 405 surfactant. This composition is heated to 40° C., applied toa sheet of poly(methyl methacrylate) [plexiglas] and dried at 40° C.

In addition to fixing histological, cytological and proteinaceouspreparations, the precoted plastic slide becomes resistant to solventssuch as xylene and toluene frequently encountered in slide preparation.

EXAMPLE 5

    ______________________________________                                        Ingredient              Amount                                                ______________________________________                                        98% pyrrolid-2-one      14.5   ml.                                            urea                    3.15   g.                                             polyethylene glycol (200 mw)                                                                          10.5   ml.                                            zinc sulfate            1.25   g.                                             ______________________________________                                    

Seven milliliters of the pyrrolidone, 1.5 g. of the urea, 0.5 g. of thezinc acetate and 3.0 ml. of the polyethylene glycol are mixed with 500ml. of distilled water.

To 100 ml. of this solution are added the following colloid protectivecomposition:

    ______________________________________                                        Dextrin (Kohdex)       2      g.                                              Surfactant (Triton 405)                                                                              .005   mg.                                             Gelvatol G 20/60       4      g.                                              Acrylamide Formulation*                                                                              6.7    ml.                                             ______________________________________                                         *prepared by dissolving 72 g. of acrylamide in 20 ml. of glycerol, 40 ml.     of acetic acid and 75 ml. of acetone.                                    

The remaining 7.5 ml. of pyrrolidone, 1.65 g. of urea, 0.75 g. of zincacetate and 7.5 ml. of polyethylene glycol are mixed with 500 ml. ofwater and to this mixture are added 5 g. of Gelvatol G 20/60 and 0.025mg. of Triton 405 surfactant.

Equal parts by weight of the first solution containing the protectivecolloid preparation and the second solution are combined with 16.7 partsby weight of milk latex coated β-UCG antigen. To slides of poly(methylmethacrylate) is applied 0.280 mg. of this mixture. The mixture is driedat 40° C. to leave an evaporative residue of the stabilized antigenwhich is stable at room temperature and will react with gonadotropichormone antibody.

What is claimed is:
 1. A diagnostic composition which comprises(a) aproteinaceous substance which is reactive in a diagnostic immunologicalreaction; and (b) as a principal active preservative component for saidproteinaceous substance, a mixture of from about 10 to about 75% byweight of pyrrolid-2-one, from about 10 to about 50% by weight ofpolyol, from about 1 to about 20% by weight of a urea and from about 1to about 10% by weight of a zinc salt of a nonoxidizing organic orinorganic acid, the relative percentages of said preservative componentingredients being selected from within the respective ranges so thattheir sum is 100% of said mixture.
 2. A composition according to claim 1wherein the proteinaceous substance is an antigen or antibody.
 3. Acomposition according to claim 1 wherein the zinc salt is zinc acetateor zinc chloride.
 4. A composition according to claim 1 wherein thepyrrolid-2-one constitutes from about 40 to about 60% by weight of saidpreservative component.
 5. A composition according to claim 1 whereinsaid polyol is a polyethylene glycol which constitutes from about 20 toabout 40% by weight of said preservative component.
 6. A compositionaccording to claim 1 wherein the urea constitutes of from about 5 to 15%by weight of said mixture.
 7. A composition according to claim 1 whereinsaid pyrrolid-2-one constitutes from about 55 to about 60% of saidpreservative component, said polyol is polyethylene glycol having amolecular weight of about 200 and constitutes from about 20 to about 25%of said preservative component, said urea constitutes from about 10 to15% of said preservative component and said zinc salt is zinc acetateand constitutes from about 3 to about 5% of said preservative component.8. An aqueous formulation of a composition according to claim 1comprising at least sufficient water to solubilize said mixture andsuspend said proteinaceous substance.
 9. A composition according toclaim 1 wherein said proteinaceous substance is bound to a carrier. 10.A diagnostic test device comprising a base strip of material which isnon-absorbent to and insoluble in water and carrying thereon at leastone deposition area in which area is carried the evaporative residue ofa diagnostic composition according to claim
 1. 11. A device according toclaim 10 wherein evaporative residues of different compositions aredeposited in a single zone of said strip.
 12. A device according toclaim 10 wherein said strip is divided into a plurality of zones, eachcarrying the evaporative residue of the same or different diagnosticcompositions.
 13. A device according to claim 10 wherein said base stripis glass.
 14. A device according to claim 10 wherein said proteinaceoussubstance is an antigen or antibody.
 15. A device according to claim 10wherein said base strip is plastic.
 16. A device according to claim 15wherein said plastic is polystyrene.
 17. A device according to claim 15wherein said plastic is a polyacrylate or polymethacrylate.
 18. A deviceaccording to claim 15 wherein said plastic is polyvinyl chloride.